The present invention relates to a novel approach to engineer, through mutagenesis and site-directed chemical conjugation, specific, well-defined dualPEGylated-protein bioconjugates, consisting of two polyethylene glycol (PEG) macromolecules chemically conjugated to the protein at two specifically defined amino acid residues. The described dualPEGylated-protein bioconjugates show substantially improved bioefficacy and biocompatibility.
Due to recent advances in genetic and cell engineering technologies, proteins known to exhibit various pharmacological actions in vivo are capable of production in large amounts for pharmaceutical applications. Such proteins include erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), interferons (alpha, beta, gamma, consensus), tumor necrosis factor binding protein (TNFbp), interleukin-1 receptor antagonist (IL-1ra), brain-derived neurotrophic factor (BDNF), kerantinocyte growth factor (KGF), stem cell factor (SCF), megakaryocyte growth differentiation factor (MGDF), osteoprotegerin (OPG), glial cell line derived neurotrophic factor (GDNF) and obesity protein (OB protein). OB protein may also be referred to herein as leptin.
Leptin is active in vivo in both ob/ob mutant mice (mice obese due to a defect in the production of the OB gene product) as well as in normal, wild type mice. The biological activity manifests itself in, among other things, weight loss. See generally, Barinaga, xe2x80x9cObesexe2x80x9d Protein Slims Mice, Science, 269:475-476 (1995) and Friedman, xe2x80x9cThe Alphabet of Weight Control,xe2x80x9d Nature, 385:119-120 (1997). It is known, for instance, that in ob/ob mutant mice, administration of leptin results in a decrease in serum insulin levels, and serum glucose levels. It is also known that administration of leptin results in a decrease in body fat. This was observed in both ob/ob mutant mice, as well as non-obese normal mice. Pelleymounter et al., Science, 269:540-543 (1995); Halaas et al., Science, 269:543-546 (1995). See also, Campfield et al., Science, 269:546-549 (1995) (Peripheral and central administration of microgram doses of leptin reduced food intake and body weight of ob/ob and diet-induced obese mice but not in db/db obese mice.) The OB protein, analogs, derivatives and use thereof as modulators for the control of weight and adiposity of animals, including mammals and humans, has been disclosed in greater detail in WO 96/05309, supra. See also, PCT International Publication Numbers WO 96/40912, WO 97/06816, 97/18833, WO 97/38014, WO 98/08512 and WO 98/28427. The OB protein, or leptin, as it is called herein, causes weight loss in humans; Greenberg et al., xe2x80x9cPreliminary safety and efficacy of recombinant methionyl human leptin (rL) administered by SC injection in lean and obese subjects.xe2x80x9d Poster presented at: 58th Annual Meeting and Scientific Sessions of the American Diabetes Association; Jun. 14, 1998; Chicago, Ill. In none of these reports have toxicities been observed, even at the highest doses.
Preliminary leptin induced weight loss experiments in animal models predict the need for a high concentration leptin formulation with chronic administration to effectively treat human obesity. Dosages in the milligram protein per kilogram body weight range, such as 0.5 or 1.0 mg/kg/day or below, are desirable for injection of therapeutically effective amounts into larger mammals, such as humans. An increase in protein concentration is thus necessary to avoid injection of large volumes, which can be uncomfortable or possibly painful to the patient.
Unfortunately, for preparations of a pharmaceutical composition for injection in humans, it has been observed that the leptin amino acid sequence is insoluble at physiologic pH at relatively high concentrations, such as above about 2 mg active protein/milliliter of liquid. The poor solubility of leptin under physiological conditions appears to contribute to the formation of leptin precipitates at the injection site in a concentration dependent manner when high dosages are administered in a low pH formulation. Associated with the observed leptin precipitates is an inflammatory response at the injection site which includes a mixed cell infiltrate characterized by the presence of eosinophils, macrophages and giant cells.
To date, there have been no reports of stable preparations of human OB protein at concentrations of at least about 2 mg/ml at physiologic pH, and further, no reports of stable concentrations of active human OB protein at least about 50 mg/ml or above. The development of leptin forms which would allow for high dosage without the aforementioned problems would be of great benefit. It is therefore one object of the present invention to provide improved forms of leptin by way of site-specific chemical modification of the protein.
There are several methods of chemical modification of useful therapeutic proteins which have been reported. For example, there is a long history of proteins chemically modified with polyethylene glycol demonstrating improved pharmacological properties. Among these properties are increased serum half-life, improved solubility and decreased immunogenicity. Chemical modification with a single 20 kDa polyethylene glycol (PEG) polymer at the N-terminus of leptin results in a highly efficacious molecule which demonstrates substantial dose reduction and increased solubility relative to the unmodified native protein; see, e.g., PCT WO 96/40912, supra, at page 8 et seq. for a description of N-terminally derivatizing leptin (therein referred to as OB Protein). Although the PEG polymer extends the circulating half-life of the bioconjugate and may impart some reduced immunogenicity, it has also been found to accumulate in kidney vacoules when administered regularly at a high dose (10 mg/kg). This phenomena has been reported with other PEGylated protein preparations; see e.g., Conover et al.,Artificial Organs, 21(5):369-378 (1997); Bendele et al., Toxicological Sciences, 42:152 (1997). Although it is not known if such vacuoles are detrimental to the health of an individual, it is preferable that drug administration have no associated anatomical abnormalities.
It was thus an object of the present invention to produce a leptin conjugate sufficiently large to escape glomerular filtration by the kidneys, and thus demonstrate little or no propensity to induce kidney vacuolation. Production of such conjugates is achieved using a course of rational mutagenesis combined with the site-directed dual PEGylation of leptin with appropriately sized polymers. Importantly, unlike the current strategies for poly-PEGylation of proteins, which result in heterogeneous mixtures of positional isoforms which are hard to separate and which vary in intrinsic bioactivity, the dualPEGylated protein bioconjugates of the present invention contain specific conjugation sites which were engineered to provide homogenous preparations which maintain the intrinsic bioactivity of the conjugate while exploiting the pharmacokinetic advantages of PEGylated-protein conjugates.
The present invention relates to substantially homogenous preparations of chemically modified proteins, e.g. leptin, and methods therefor. Unexpectedly, site-specific chemical modification of leptin demonstrated advantages in bioavailibility and biocompatibility which are not seen in other leptin species. Importantly, the methods described herein are broadly applicable to other proteins (or analogs thereof), as well as leptin. Thus, as described below in more detail, the present invention has a number of aspects relating to chemically modifying proteins (or analogs thereof) as well as specific modifications of specific proteins.
In one aspect, the present invention relates to a substantially homogenous preparation of dualPEGylated-leptin (or analog thereof) and related methods. Importantly, the method described results in a high yield of dualPEGylated protein which is modified exclusively at two defined sites, thereby providing processing advantages as compared to other species involving random modification. The present invention stems from the observation that, as compared to unaltered native recombinant human leptin, dualPEGylated-recombinant human leptin has substantially improved bioactivity and biocompatibility.
It has been found, surprisingly and importantly, that dualPEGylated-leptin bioconjugates prepared from 20 kDa, 30 kDa, and 40 kDa PEG polymers, proved highly efficacious, and demonstrated little or no propensity for kidney vacuolation. Significantly, when the dualPEGylated-leptin bioconjugates were administered in a single dose, weight loss was maintained for over 7 days, at twice the level of an equivalent dose of unmodified leptin dosed daily over the 7 day period.
The recombinant human leptin used in the working examples below was first modified such that select cysteine mutations were engineered into the leptin protein sequence. The resultant recombinant human leptin analogs were recoverable in high yield and then used to prepare the dualPEGylated bioconjugates. Thus, in one aspect, the present invention relates to human leptin having cysteine mutations engineered into positions 72 or 78 of the leptin protein sequence.
The present invention also relates to dualPEGylated human leptin bioconjugates wherein PEG is conjugated at the N-terminus and at position 78 of the leptin protein sequence. Preferably the PEG has a molecular weight from about 10 kDa to about 100 kDa. A particularly preferred PEG is about 20 kDa for each polymer chain.
The present invention further relates to all of the dualPEGylated human leptin bioconjugates as above, in a pharmaceutically acceptable carrier.
The present invention further relates to processes for preparing dualPEGylated protein bioconjugates as above. The principal embodiment of the method for making the substantially homogenous preparation of dualPEGylated-protein comprises: (a) engineering a cysteine residue into a specific amino acid position within the amino acid sequence of said protein to provide an analog of said protein; (b) conjugating a polyethylene glycol to said analog at said cysteine residue to provide a monoPEGylated protein conjugate; (c) conjugating a second polyethylene glycol to the N-terminus of said conjugate to provide a dualPEGylated bioconjugate; and (d) isolating said dualPEGylated bioconjugate.
The present invention also relates to methods of treatment of individuals using dualPEGylated human leptin bioconjugates as above.